International Journal of Applied Research 2016;2(8): 19-24 ISSN Print: 2 394-7500 ISSN Online: 2394-5869 Impact Factor: 5.2 Haemocytes count of Poekilocerus pictus (Fabr.) IJAR 2016; 2(8): 19-24 www.allresearchjournal.com (Orthoptera: Acrididae) during fungal infection Received: 04-06-2016 Accepted: 05-07-2016 Namita Jain, Janak Ahi Namita Jain Department of Zoology Dr. H. S. Gour University Abstract Sagar, Madhya Pradesh, India Insect circulating haemocytes are primarily responsible for the immune defense against parasites and pathogens. In this paper THC and DHC after Aspergillus niger infection were elucidated. Injection of Janak Ahi A. niger conidia into P. pictus resulted in changes in the total haemocyte count and differential Department of Zoology haemocytes counts. Total haemocyte counts were higher at 2h and 4h post infection and lower at 8h Dr. H. S. Gour University and 24h post infection than control insects. There was a considerable change in the relative percentage Sagar, Madhya Pradesh, India of granulocytes and plasmatocytes in the hemolymph after challenge with A. niger. It is observed that plasmatocytes and granulocytes are the principal cell types, which respond the most during the defense. Keywords: THC, DHC, Poekilocerus pictusand Aspergillus niger. Introduction Insects represent the largest class within the animal kingdom in terms of species number. The resistance of insects to pathogens has certainly contributed to their extreme proliferation and diversity. At present, insects are found in most of the biological niches except for the deep marine environment and the polar regions. More than one million of insect species have been described and it is estimated that an equivalent number of species remains to be identified. Multicellular animals defend themselves against infectious organisms by two systems known as innate and acquired immunity. The innate immune system relies on germline encoded factors for recognition and killing of foreign invaders, whereas the acquired immune system produces receptors by somatic gene rearrangement that recognize specific antigens and that [11] allow organisms to develop an immunological memory (Fearon, 1997) . Insects lack an acquired immune system but have a well-developed innate response. Initial defences include the physical barriers of the integument or gut, clotting responses by hemolymph, and the production of various cytotoxic molecules at the site of wounding. Foreign entities that pass these barriers and enter the hemocoel must contend with additional cytotoxic molecules as well as an array of different haemocytes. The insect immune system is further subdivided into humoral and cellular defence responses. Humoral defences include the production of antimicrobial peptides (Meister et al., 2000; Lowenberger, 2001)[26, 25], reactive intermediates of oxygen or nitrogen (Bogdan et al., 2000; Vass and Nappi, 2001)[4], and the complex enzymatic cascades that regulate coagulation or melanisation of hemolymph (Muta and [27, 13] Iwanaga, 1996; Gillespie et al., 1997) . In contrast, cellular defense refers to haemocyte- mediated immune responses like phagocytosis, nodulation and encapsulation (Strand and Pech, 1995; Schmidt et al., 2001) [35, 31]. While great progress has been made over the last several years in identifying antimicrobial peptides and the signaling pathways that regulate their synthesis, much less is known about control of cellular defense responses. This is due in large part to the small size of many insects, which makes collection of haemocytes and identification of haemocyte-produced effector molecules difficult. It is also often difficult to conduct manipulative experiments on haemocyte-mediated defense responses in vivo or to isolate defined populations of haemocytes for use in experiments conducted in vitro. The main effectors of cellular immune responses in insects are the blood cells or haemocytes. Correspondence Namita Jain Many studies have been made of haemocyte counts, either as the total haemocyte count Department of Zoology (THC) or differentially according to type (DHC). It is well attested that both THC and DHC Dr. H. S. Gour University change rapidly during immune responses to infection (Lackie, 1988) [24]. Taubereager Sagar,Madhya Pradesh, India (1935,1936)[36, 37] and Beard (1945) [3] noted an increase in THC ~19~ International Journal of Applied Research in the hemolymph of insects due to bacterial infection. But Method of Injection many authors have observed drastic reduction in the number Injection of fungus Aspergillus niger for THC and DHC of haemocytes during bacterial infections (Kostritsky et al studies 1924; Babers, 1938; Wittig, 1965, Krishnan and Chaudhuri, Insects were surface sterilized by swabbing their surfaces 1998, 1999; Krishnan et al., 2000)[20, 2, 21, 22, 23]. In bacterial- with 70% ethanol. Control insect were injected with 10 l of infected Prodenia larvae, haemocytes counts either sabouraud dextrose broth and test insects were infected by remained unchanged or declined markedly; thus injecting a standard fungal dosage of 105 CFU/insect, fungus Rosenberger and Jones (1960)[30] concluded that the Aspergillus niger were injected in 10l aliquots, using a 25 haemocytes are not very effective in protecting the insect. In l Hamilton syringe Gallaria, THC increased upto 48 hours following infection with Staphylococcus and decreased rapidly (Werner and Collection of hemolymph for THC and DHC studies Jones, 1969)[38]. High doses of infection in Pseudoletia For the counting of the total haemocyte count (THC), the larvae resulted in drastic reduction in plasmatocytes and a arthropodial membrane of the legs was first swabbed with great increase in spherulocytes and prohaemocytes, with no 70% ethanol, allowed to air dry and then pierced with a change in the number of granulocytes (Wittig, 1966)[40]. sterile needle and hemolymph was collected and diluted 20 Low doses, on the other hand, had a different effect on the times in saline versene (NaCl-.9g KCl-.942g CaCl2 -.082g blood picture because the granulocytes and plasmatocytes NaHCO3 -.002g D.W.- 100ml + 2% versene) and transferred decreased while the spherulocytes and prohaemocytes immediately to an improved Neubauer haemocytometer. increased significantly (Wittig, 1966)[40]. Krishnan and The number of cells were counted under the Olympus light Chaudhuri (1998; 1999)[21, 22] and Krishnan et al (2000)[23] microscope and calculated by the formula suggested by observed a reduction in both plasmatocytes and granular Jones (1962)[19]. THC was done at 2h, 4h, 8h and cells of B. mori infected with bacteria. 24hpostinfection in bacterial infected Poekilocerus pictus Poekilocerus pictus (Fabr.) is an orthopteran insect known and fungus infected Poekilocerus pictus. as a monophagous pest of the medicinal plant ak (Calotropis For the counting of the relative number of different types of sp.). Its large size makes this insect easy to manipulate haemocytes (differential haemocyte count, DHC), the physically, enabling easy injection of substances. Another arthropodial membrane of the legs was first swabbed with advantage to the large size of P. pictus is the ability to 70% ethanol, allowed to air dry and then pierced with a collect between 0.5-1 ml of haemolymph from each insect sterile needle and haemolymph was collected by bleeding a meaning that fewer insects are required for experiments. drop of haemolymph onto a glass microscope slide then In the present study chief defensive cells involved in celluler expelled onto a glass microscope slide. The slide was then immune response against Aspergillus niger infection was incubated for 10 min at room temperature to allow the elucidated. As notmuch work has been done on P. pictus haemocytes to adhere to the slide. The glass slide with the regarding insect immunity against bacterial and Aspergillus air dried film were immersed in Giemsa solution for 20 niger infection. The more detailed knowledge of minutes to 2 hours (1drop of concentrate per milliliter distill Poekilocerus pictus defense response will help us to achieve water) and then mounted. The haemocytes were observed at greater success in our efforts for biological control of insect 400X magnification using an Olympus light microscope. population and to explore insect immunity in general. Infact Each time 100 cells were counted and percentage of various this study encompasses comprehensive investigation into haemocytes was determined. The method of Shapiro (1966) immune response of P. pictus which will help to clarify, [32] was used to count relative number of different types of some of unresolved issues in insect immunity against haemocytes. DHC was done at 2h, 4h, 8h and Aspergillus niger infection with respect to the chief 24hpostinfection in bacterial and fungal infected haemocytes involved in defense response and the insight Poekilocerus pictus. The experiment was repeated five times into basis and mechanism of defense reactions in using completely random design (CRD). Data were Poekilocerus pictus. expressed as the mean ± standard error of mean. Materials and methods Statistical analysis Insect collection and its rearing The data were expressed as mean ± Standard error. Grasshoper, Poekilocerus pictus (Orthoptera: Acrididae) Statistical analysis of data obtained from the THC and DHC were collected from Calotropis plants from different were analyzed through one way analysis of variance locations around Sagar (M.P.). They were maintained under (ANOVA) and Dunnet multiple comparison